I am calling out all who think they have good MiSeq runs. Gaze at the perfection of this run and weep at the inadequacy of your wretched instruments:

But seriously...
Accidently clustered at a higher density than normal. But the results were still good. Raw cluster density was 1154 Kclusters/mm^2. 8.8 billion bases total. 6.8 billion bases >Q30.

Kind of interesting, this indicates that 0.6x0.4 = 24% of read pairs have zero sequencing errors in them!

And, just to add historical perspective, this is around the amount of data generated by 13 GS-FLX runs. (And we would bill out a GS-FLX run at 5x the cost of a MiSeq run.) Also, at 80 kb per 3730XL run, we would need 13,000 of them to equal the output of this single MiSeq run. That is 3 years of non-stop runs on a 3730XL!

I mentioned in other thread that I was getting decent results at density of as much as 1500. My problems is associated more with short libraries made from ancient DNA, so pushing longer than 2x151 cycles makes little gains. But with density of 1100 I got about 75% of >30Q with 2x151 cycles.

We have not had any problems getting the clusters to register without spiking in phiX with 16S runs. It is the quality values assigned to the bases (in latter half of 2 x 250 bp runs), where the problem occurs. Sequences being generated have been acceptable for the end users with no problems about the actual base calls.

Would be curious to see how you fare with your 16S runs. I assume you will be spiking in phiX.

We have not had any problems getting the clusters to register without spiking in phiX with 16S runs. It is the quality values assigned to the bases (in latter half of 2 x 250 bp runs), where the problem occurs. Sequences being generated have been acceptable for the end users with no problems about the actual base calls.

Would be curious to see how you fare with your 16S runs. I assume you will be spiking in phiX.

We will likely spike in genomics libraries of some sort. That way some useful data is generated from the genomic 'ballast' library.

So you are saying that the quality values assigned are inaccurate (lower than they should be)?

We should be getting back our MiSeq 16s results within the next day or two- did a 50% spike-in of gDNA under the recommendation of the center. Right now our SOP is to find some genome in our lab that needs sequencing and use that to up the diversity on the run. Otherwise it's 90 barcoded samples...I'll let you guys know how it went when we get it!

Was the incredible amount of data you got from one run using an upgraded MiSeq? We have not had the upgrade yet. Our record with RNAseq libraries has been 2.5 Gb of calls >Q30 (1781 K/mm2, 78.5% pass, 13.37 M raw reads, 2x150bp). RNAseq libraries allow higher resolvable cluster density than Nextera yeast genome libraries. Our record with Nextera libraries is 2.4 Gb of calls >Q30 (1262 K/mm2, 90.1% pass, 9.98 M raw reads, 2x150bp).

We suspect that the higher cluster density with RNAseq libs is due to there being fewer longer fragments in the library, which would presumably form larger clusters that would be harder to resolve. We would love to know about tricks to achieve higher cluster densities with Nextera libraries. We already modified the PCR in the protocol by reducing the extension time from 3 min to 1 min and adding two more cycles (7 total).

Was the incredible amount of data you got from one run using an upgraded MiSeq?

Yes.

Quote:

Originally Posted by creeves

We suspect that the higher cluster density with RNAseq libs is due to there being fewer longer fragments in the library, which would presumably form larger clusters that would be harder to resolve. We would love to know about tricks to achieve higher cluster densities with Nextera libraries. We already modified the PCR in the protocol by reducing the extension time from 3 min to 1 min and adding two more cycles (7 total).

You want to remove the larger fragments, you mean? You could do an Ampure upper cut, right?

I am finally running of my bacterial pooled DNA library (which I made using Nextera XT) on the Miseq using MiSeq V3. I have checked the machine now and it shows that after 70 cycles, the >=Q30 is 96%, cluster passing filter 91.4%, and the cluster density is 1092K/mm.

I am finally running of my bacterial pooled DNA library (which I made using Nextera XT) on the Miseq using MiSeq V3. I have checked the machine now and it shows that after 70 cycles, the >=Q30 is 96%, cluster passing filter 91.4%, and the cluster density is 1092K/mm.

The Q30 is now 83.1% after 250 cycles, yesterday it was 96% after 70 cycles. But rest of the parameters are consistent: clusters PF 91.4%, cluster density 1092K/mm^2, which are the same as yesterday. Do you speculate anything? DO you think that the run will be okay?